Abstracts

Rationale: Hypoxia modifies GABA_A receptor (GABAR) function and can cause seizures, encephalopathy, or myoclonus. Our previous studies in cortical neuron culture have shown that hypoxic exposure (1% O₂ for 4 h) resulted in downregulation of maximal GABA current at 0 h and 48 h recovery without changing GABA EC₅₀. We wished to determine whether the effects of hypoxia on GABA receptor function were related to calcium entry via L-type voltage gated calcium channels (L-VGCC). Methods: To characterize the effects of hypoxia on neuronal GABARs, we exposed rat cortical neurons 13-15 DIV to 1% O₂ for 4 h, followed by recovery times of 0 h to 48 h, and used whole-cell and gramicidin perforated-patch clamp recording to assess GABAR currents and pharmacology. To evaluate whether the effects of hypoxia on cortical neuron GABA currents were due to calcium (Ca²⁺) entry through L-type voltage-gated calcium channels (L-VGCCs), we co-incubated cortical neuron cultures with the L-VGCC blocker, nitrendipine (NT, 3 µM), during 4 h control or hypoxic exposure, with subsequent washout and replacement with fresh neuronal culture medium. Results: Whole-cell currents elicited by GABA after incubation with NT (3 µM, 4 h) were significantly larger than control currents from untreated neurons (control: 4289 ± 459 pA, n=10; control + NT: 6341 ± 484 pA, n=11, p<0.05) without a change in EC₅₀ or Hill slope. In cells recorded at 0 h, 24 h or 48 h after 4 h hypoxia in the presence of NT, there was no significant change in maximal current, EC₅₀ value or Hill slope from control cells (exposed to NT) (H4 + NT, R0: 6748 ± 820 pA, n=8; H4 + NT, R24: 5409 ± 945 pA, n=8; H4 + NT, R48: 6212 ± 524 pA, n=9; p>0.05). Maximal current values at each post-hypoxic time point were significantly larger than those recorded at these time points after 4 h hypoxia in the absence of NT. NT during hypoxic exposure prevented the increase in zolpidem potentiation of GABA currents after 48 h recovery (control + NT: 269.4 ± 18.7%; H4 + NT, R48: 194.0 ± 16.3%, p<0.05). NT exposure also blocked a hypoxia-induced depolarizing shift in Cl^- reversal potential 24 h after hypoxia. The Cl^- reversal potential at 24 h and 48 h recovery after 4 h hypoxia in the presence of NT did not change compared with control (control + NT: -50.41 ± 3.59 mV, n=7; H4 + NT, R24: -52.93 ± 4.36 mV, n=6; H4 + NT, R48: -51.77 ± 4.17 mV, n=6, p>0.05). Conclusions: The effects of hypoxia on maximal GABAR currents, zolpidem pharmacology and Cl^- reversal potential thus require depolarization-induced Ca²⁺ entry via L-VGCCs, and constitutive L-VGCC activity appears to reduce maximal GABAR currents in control neurons via a Ca²⁺-dependent process. Ca²⁺-dependent modulation of GABAR currents via L-VGCCs may be a fundamental regulatory mechanism for GABAR function.